Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1’s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

This manuscript describes experiments on the gafing of Piezo ion channels in pressurized membrane patches.In parficular, data is presented that indicates that the micropipefte diameter and opening angle, as well as cytoskeletal organizafion, can affect Piezo gafing.The authors also used a confinuum elasfic model to make statements about how the membrane stress might distribute in the aspirated membrane patch.The most interesfing aspect of the paper is, perhaps, the observed dependence of F-acfin accumulafion on the fip angle.
While the general issue of how constraints on the membrane affect the gafing of Piezo ion channels is fimely and interesfing, the experiments presented here only offer very qualitafive insights.Furthermore, the modeling presented here is both too detailed and too idealized to provide any real insight---it is too detailed in the sense that it involves many effecfively free parameters that make it impossible to make any definite predicfions, while it is too idealized in the sense that it does not seem to correctly capture the physics of membrane tension propagafion and Piezo gafing relevant for the specific experiments described here.
-In their modeling, the authors put much emphasis on the von Mises stress distribufion in the membrane, which raises several issues: (1) The authors generally only show normalized stresses, from which it is not clear that the results even pass a basic sanity check---generically, lipid bilayers can only support stresses of a few percent.(2) There are two different kinds of membrane tension---the "mechanical" membrane tension the authors (implicitly) consider here and membrane tension connected to local membrane bending/unbending, which does not involve a change in membrane area.The lafter kind of membrane tension tends to be energefically much less costly and is most relevant for membranes that show roughness due to, e.g., thermal fluctuafions, heterogeneous lipid composifions, protein-induced membrane deformafions, or membrane-cytoskeletal interacfions.It is this lafter kind of membrane tension that seems to underlie Piezo gafing, with Piezo being curved in its closed state and Piezo unbending during gafing.The mechanical membrane tension focused on by the authors does not seem to be the relevant membrane tension to consider here.(3) Given experimental and modeling uncertainfies, it is unclear what the precise connecfion between the von Mises stresses calculated here and the (mechanical) membrane tension might be.(4) The distribufion of membrane tension over the scale of microns considered by the authors will be strongly affected by membrane composifion---in parficular, the presence and organizafion of membrane proteins---as well as membrane-micropipefte interacfions.It is unclear why the stress distribufions calculated by the authors should have any relevance to the membranes studied experimentally.
-It has been known for some fime that Piezo gafing is affected by force on the membrane/the channel and cytoskeletal aftachments (e.g., ref. 26 in the manuscript and Nature Communicafions 7.1 (2016): 12939---a paper that should be discussed in this context).The present manuscript adds a twist to this, by pufting emphasis on the micropipefte shape.To gain new, solid understanding of Piezo gafing in cell membranes, quanfitafive data on the molecular mechanisms underlying the observed behavior would be required.For instance, there are many ways in which the micropipefte might affect membrane tension---e.g., through the physics of membrane-pipefte adhesion, the constrained geometry at the fip of the micropipefte, patch-clamp induced changes in membrane organizafion, etc.Furthermore, in addifion to changes in the local membrane tension, Piezo gafing is also affected by the local membrane geometry---see, e.g., eLife 7 (2018): e41968---and it is not clear whether the results in the present manuscript are due to changes in the local membrane tension (as suggested by the authors) or due to the local membrane geometry (or a combinafion of both).
-Especially in the discussion secfion, the manuscript makes strong claims that are not sufficiently substanfiated by the data or the modeling, given their very qualitafive nature, thus overselling the results of this study.Moreover, some of the language used here, starfing with the term "buckle" in the fitle, is rather sloppy; "Buckling" has a well-defined meaning in science, and the paper does not provide much evidence that this is indeed what is going on here.Also, the paper starts off by talking about the binding site-driven or membrane curvature-driven Piezo localizafion described in refs.19, 20 in the manuscript, while the manuscript argues (based on somewhat flimsy evidence) that the observed effects are driven by changes in the local membrane tension.This is confusing, as membrane geometry, Piezo binding, and local changes in membrane tension provide disfinct physical mechanisms potenfially affecfing Piezo gafing (see also the point above).

Summary
In this manuscript, Wang et al develop combine mulfi-channel imaging and micropipefte aspirafion to study how local membrane deformafion acfivates the mechanically acfivated ion channel Piezo1.
Combining this approach with computafional analysis to calculate tension changes at the deformed membranes, the authors find that local Piezo1 responses scale with the pipefte diameter and fip angle.Moreover, they discover a rapid acfin redistribufion into a buckle in the region of the cell right outside the pipefte fip.This redistribufion is in part Piezo1-dependent and promotes further channel acfivafion.Importantly, contracfility is dispensable for acfin buckle amplificafion of Piezo1 funcfion, poinfing at acfin accumulafion as a mechanical input by itself.
The arficle is clearly wriften and illustrated, and its solid approach tackles an important issue regarding a channel family aftracfing a lot of aftenfion in recent years: how are these channels acfivated by the cell environment?These findings seem especially relevant for RBC deformafion by vessels and downstream volume regulafion, known to depend heavily on Piezo1.However, the same could be said for white blood cells, and metastafic cancer cells both during blood-borne dispersion or invasion.In addifion, one of the answers of this paper has relevant consequences for us Piezo researchers: we need to consider how we are polishing and holding our patch clamp pipeftes in our daily experiments.These examples signify the broad relevance of this work Overall, I think this paper should be published after addressing some comments.

Minor comments
• This is clearly out of the scope of this paper and should not affect any editorial decision.Just curiosity: do the authors have any experiment using GOF Piezo1 mutants known to control RBC volume and cause xerocytosis (Cahalan, eLife, 2015 and references therein)?Is their method capable of capturing these differences?Are the mutafions shifting any of the curves: diameter, angle?What happens to acfin in those cells?
• Acfin buckle formafion requires Piezo1.Describing the mechanism linking Piezo1 to Arp2/3-dependent F-acfin (based on the CK666 experiments) seems out of the scope of the paper.However, the fact that Piezo1 triggers its acfin-based own amplifier seems very relevant and should be discussed more in detail.
• Figure 1A shows nucleated cells and RBCs in the cross-secfion of the vessel (top right) but seems to focus on nucleated cells in the longitudinal cut (boftom left).However, most experiments use RBCs (it's one of the paper keywords!).Just showing RBCs would make things clearer.
• Line 240-241: If I got this right, which further substanfiafing should read which further substanfiated or suppress which.
• I think the fitle of Fig. S3 needs rewrifing.

Dear Reviewers,
We express our gratitude for the thoughtful critiques and constructive comments, which have been pivotal in enhancing the manuscript's clarity and significance.For the past three months, we have undertaken comprehensive revisions, performed new experiments and computational modeling in response to the feedback, ensuring that each point raised has been meticulously addressed.The major revision and changes are outlined as below:  Aspirated cell membrane tension mapping through Fluorescence Lifetime Imaging Microscopy (FLIM): We set up the concurrent FLIM imaging with our fluorescent micropipette aspiration assays (fMPA) and devised a new protocol using Flipper-TR ® -Live cell membrane tension probe to map the tension distribution from the aspirated red blood cell's tongue to body (revised Fig. 3d-h and Fig. R3).
 Elastic shell finite element analysis (FEA) to simulate red blood cell aspiration by micropipettes with various tip angles: We applied new FEA analysis using elastic shell framework and rationale.The simulated maximum principal stress distribution in the aspirated tongue well correlated with the experimental membrane tension mapping by FLIM (revised Fig. 3a-c and Figs.R1 and 2)  Article Title Revised: "Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution"  Abstract Revision: "...increased micropipette tip angles intensify PIEZO1 activation, linked to a significant reorganization of F-actin, resulting in physical constrictions that amplify the channel's activity in regions undergoing tension stress…"  Introduction Update: "The inherent curvature of PIEZO1 trimers conveys sensitivity to tension, further influenced by their distinct architecture and size.PIEZO1's role in mechanosensing is highlighted by its preferential localization within the biconcave dimples of red blood cells, crucial for erythrocyte function."  Figures Adjusted: o  Response: Upon reflection, we acknowledge the concerns regarding the qualitative nature of our findings and the potentially abstract nature of our modeling.In response, we have: 1.
Refined our computational model using an updated inverse finite element analysis (FEA) technique to determine the material parameters for creating an elastic shell model.This has allowed us to more accurately simulate the biomechanical behavior of RBCs under differential micropipette aspiration angles (θ = 0°, 5°, and 10°), providing a more realistic representation of the tension dynamics post-aspiration.As a result, the updated model correlates with experimental observations of RBC deformation closely, enhancing the predictive power of our simulations.

2.
Augmented our methodology with a comprehensive Fluorescence Lifetime Imaging Microscopy (FLIM) study.Employing a membrane tension-sensitive probe, Flipper-TR® (Colom, Derivery et al. 2018), we have quantified the distribution of membrane tension across RBCs subject to micropipette aspiration.This has more precisely elucidated the relationship between micropipette tip angle and PIEZO1-mediated calcium mobilization, affirming our hypothesis that membrane tension is a critical factor in physical constriction induced PIEZO1 activities.
-In their modeling, the authors put much emphasis on the von Mises stress distribution in the membrane, which raises several issues: (1) The authors generally only show normalized stresses, from which it is not clear that the results even pass a basic sanity check---generically, lipid bilayers can only support stresses of a few percent.

Response:
We appreciate the reviewer's critical insights on the presentation of von Mises stress distributions in our preliminary modeling work.To clarify, we have refined our FEA approach in the following ways: 1.
We have calibrated our cell aspiration modeling parameters against the real experiments to ensure the physiologically relevance (Fig. R1).Specifically, we have imposed constraints on the stress values to reflect the limited stress capacity of lipid bilayers, addressing the sanity check concerns.Action taken: 1.
The Method section in the revised manuscript has been updated with the new section 'Elastic shell finite element analysis on single cell micropipette aspiration': Page 29 line 13 -Page 31 line 12: 'The simulation of micropipette aspiration was conducted using LS-DYNA (ANSYS, US), a commercially available finite element method (FEA) analysis software.RBCs were considered as an elastic shell with thickness of 200 nm to ensure deformability during simulation (Fig. S13a).The material was considered isotropic and thus, the stress-strain relationship was expressed as: where σ and ε are the three dimensional stress and strain tensor, respectively (Bower 2009).
The stress can be written as: The fourth-order elasticity tenor C can be simplified as: where E is the Young's modulus (120 Pa), υ is the Poisson's ratio (0.49).During the initial stage of simulation, RBC geometry was considered based on a continuous degree-4 surface which is constrained by three principal dimensions: the main diameter d, the thickness at the dimple centre, b, and the maximum thickness, h (Kuchel, Cox et al. 2021).The geometry is reconstructed based on the equation: where, The geometrical constraints are considered to be d = 8, b = 1, and h = 2.12 μm (Fig. S12a).
Before performing stress analysis, we utilized the inverse FEA to obtain the material properties of the human RBC.Thereby, we measured the deformation of the RBC aspirated by the θ = 0° (Fig. S13b) and then evaluated the material properties of the aspirated cell inversely.The force applied to the RBC was considered as ∆p = -25 mmHg which was corresponded to the experiment results, while the aspirated tongue length in the simulation correlated with the experiment measurement to derive the material properties (E and υ).After obtaining the material properties, we confirmed that the tongue protrusion length was well aligned with the experiment measurements (Fig. S13c) and thereby, the extracted material parameters and protrusion length were then implemented to the simulation at tip angle θ = 0°, 5°, and 10° as boundary condition.Of note, an aspirating pressure ∆p = -25 mmHg was always applied to the nodes at the aspirated dome during the explicit dynamic analysis.
Since the micropipette is significantly stiffer than red blood cell, the glass micropipette was considered as a rigid body.A fillet radius was applied to the tip of the micropipette to mimic the experimental setups and avoid high element distortion and contact singularity.The frictionless boundary condition was applied to the cell and the adjacent micropipette wall since BSA was included during aspiration experiments.Pure penalty boundary was applied to the contact between the micropipette and the cell.while the body was fixed when mechanical anchor was applied.In order to compare the results, maximum principal stress was calculated for each studied case (Bavi, Nakayama et al. 2014) that can be calculated from The rest are the shear stress during deformation.The values reflected the trend of membrane tension elevation due to the aspiration (Bavi, Nakayama et al. 2014) and were normalized based on the maximum value of all conditions at each cell type." Meanwhile, to avoid the confusion raised by the wording, we redefine the stress change we found here as the "normalized change of membrane tension from the resting state".The details are below: 2.
In the revised figures (Figs. 3a-c), we present the recalibrated stress distribution with explicit mention of the physiological limits to which lipid bilayers are subjected, thus ensuring the results are within a credible range.

3.
The manuscript text has been updated to explicitly state that the maximum principal stress values are normalized to the maximum stress supported by lipid bilayers, providing a clear reference frame for the reported data.
(2) There are two different kinds of membrane tension---the "mechanical" membrane tension the authors (implicitly) consider here and membrane tension connected to local membrane bending/unbending, which does not involve a change in membrane area.The latter kind of membrane tension tends to be energetically much less costly and is most relevant for membranes that show roughness due to, e.g., thermal fluctuations, heterogeneous lipid compositions, protein-induced membrane deformations, or membrane-cytoskeletal interactions.It is this latter kind of membrane tension that seems to underlie Piezo gating, with Piezo being curved in its closed state and Piezo unbending during gating.The mechanical membrane tension focused on by the authors does not seem to be the relevant membrane tension to consider here.

Response:
We thank the reviewer for the insightful comments.We agree that local membrane bending/unbending is one of the primary factors that drive the PIEZO1 gating.However, studying the membrane roughness and delving into how local membrane shape changes, particularly curvature changes at the scale of <50 nm (Ridone, Grage et al. 2018), is not the focus of this study.Here, we used the 'force-from-lipid' model to explain how the membrane tension mediates PIEZO1 ion channel activation, thus modulating calcium mobilization in the aspirated cells.Importantly, this 'force-from-lipid' model has been considered by multiple research groups in this field as one of the well-recognized models to elucidate the PIEZO1 activity under mechanical force stimulation (Young, Lewis et al. 2022, Bavi, Cox et al. 2023).
Moreover, we have acknowledged that the local membrane tension (i.e., membrane footprint and local curvature) also mediate the PIEZO1 activity (Haselwandter and MacKinnon 2018).
However, it's essential to emphasize that the local membrane bending and macroscopic cell deformation (representing two distinct types of induced tension) do not operate independently; rather, they are closely intertwined on numerous levels (Rangamani, Mandadap et al. 2014, Shi and Baumgart 2015, Golani, Ariotti et al. 2019).Again, given that the aim of this study is to explore the impact of 'physical constriction' on PIEZO1 gating and activity, we will focus on the macroscopic deformation of the cells under micropipette aspiration , a scenario where 'force-from-lipid' model is predominantly applied.

Action taken:
To make it clear, we have incorporated a section to discuss the potential contributions of membrane tension, local curvature, composition, and bonded proteins to the PIEZO1-mediated calcium mobilization observed in this study: Page 20 Line 9-33 "Our combined FLIM tension mapping and elastic shell FEA simulations demonstrate that increased membrane tension, particularly around the aspirated cell's neck region, aligns with the observed upsurge in PIEZO1-mediated calcium influx.This supports the hypothesis that microscale geometrical determinant, i.e. micropipette tip angle and subsequent physical constrictions are critical in modulating mechanosensitive ion channel PIEZO1's mechano-gating."…… "In the meantime, the induction of membrane curvature by PIEZO1 provides a potential physical determinant for its subcellular distribution.PIEZO1 has been observed to display a polarized distribution in migrating keratinocytes to be enriched at focal adhesion sites (Yao, Tijore et al. 2022), and to preferentially locate within the biconcave dimples of RBCs (Vaisey, Banerjee et al. 2022), emphasizing its significant role in RBC mechanosensing, a crucial aspect of erythrocyte function.Studies have reported that PIEZO1 activities are not only mediated by the local membrane tension (Bavi, Cox et al. 2023), but also influenced by the local membrane footprint, or membrane curvature (Haselwandter andMacKinnon 2018, Young, Lewis et al. 2022).More importantly, the resting membrane tension before any mechanical stimulation (Lewis and Grandl 2015), the cluster and density of the channels (Lewis and Grandl 2021) can also alters the sensitivity and activities of the channels.
Therefore, it is also important to take these factors into consideration in the further study when untacking the microgeometry effect on single cell mechanosensing." (3) Given experimental and modeling uncertainties, it is unclear what the precise connection between the von Mises stresses calculated here and the (mechanical) membrane tension might be.( 4) The distribution of membrane tension over the scale of microns considered by the authors will be strongly affected by membrane composition---in particular, the presence and organization of membrane proteins---as well as membrane-micropipette interactions.It is unclear why the stress distributions calculated by the authors should have any relevance to the membranes studied experimentally.
Response: Although local membrane geometry modulation is not investigated in this study, we have attempted to quantify the change of local membrane tension in the aspirated cell by performing the Fluorescence Lifetime Imaging Microscopy (FLIM) assay.In this assay, we used the mechanical tension-dependent Flipper-TR probe (Colom, Derivery et al. 2018), which responds to the push-and-pull effect in the lipid bilayer when the membrane experiences tension, and planarizes to exhibit an increased fluorescence lifetime (τ, Fig. R3) when the membrane tension is elevated.The FLIM images demonstrated that an increased tip angle θ promotes a rapid membrane tension escalation, specifically in the aspirated cell's tongue region (Figs.R1 a-c).As a result, higher PIEZO1 activities and Ca 2+ mobilization was observed (proven by the fMPA in the main manuscript).Thus, aspirating cell membrane can escalate local membrane tension, indicated by an increasing fluorescence lifetime (Fig. R3d).We have added the following figure in the main manuscript: ).Since there is a linear correlation between the fluorescence lifetime and the membrane tension (Colom, Derivery et al. 2018), our results provide clear evidence that increasing the tip angle can rapidly increase the membrane tension.Intriguingly, the FLIM results also demonstrate that the membrane tension in the body is slightly higher than the tongue when the cell is aspirated by micropipette with θ = 0° (Fig. R3e; ratio = 0.98±0.01).This tension distribution shifted when the tip angle increased (ratio = 1.08±0.01at θ = 5°), and eventually, the tongue demonstrates a much higher tension when θ = 10° (ratio = 1.14±0.01).These FLIM measurements reveal a well-aligned trend with our fMPA results (Fig. R4a; r = 0.83, p-value = 0.0429) and previous mentioned simulation in the aspirated tongue (c.f.Fig. R2a-c  Moreover, our revised elastic shell FEA modeling now incorporates a refined calculation of maximum principal stress (Bavi, Nakayama et al. 2014) to more accurately represent the mechanical stress distribution within the membrane, considering the macroscopic forces applied during micropipette aspiration.This revised approach enhances the relevance of our model to the physical conditions experienced by the cell.
Notably, our simulation results explained a much higher membrane tension will be generated at the aspirated cell tongue (Figs.3a-c), which is further supported by the FLIM tension measurement.Since elevating membrane tension is well-known as one of the major causes to the PIEZO1 gating under mechanical stimulation (Cox, Bavi et al. 2019, Young, Lewis et al. 2022), our finding imposed that increasing tip angle would be the major cause of the elevated calcium influx during aspiration, especially in the tongue.to examine the membrane tension of the RBCs by using tension-sensitive probe, Flipper-TR ® 34 (Figs.3a-c).This tension imaging probe responds to the push-and-pull effect in the lipid bilayer when the membrane experiences tension and planarize to exhibit an increased fluorescence lifetime τ when the membrane tension is elevated.As the result, it is clear that aspirating cells can increase the membrane tension, indicated by an increasing fluorescence lifetime (Fig. 3d).More importantly, It is noteworthy that when the tip angle increases, the Intriguingly, the FLIM results also demonstrate that the membrane tension in the body is slightly higher than the tongue when the cell is aspirated by a θ = 0° micropipette (Fig. 3e; ratio = 0.98±0.01).This tension distribution shifted when the tip angle increased (ratio = 1.08±0.01at θ = 5°), and eventually, the tongue experienced a higher tension when θ = 10° (ratio = 1.14±0.01)."Delving into the detailed gating mechanism of PIEZO1 on the magnitude of <50 nm curvature change is out of the scope of this study (Ridone, Grage et al. 2018).Furthermore, fMPA in this study differs from the traditional patch clamp, in which membrane-pipette adhesion is preferred to form a giga seal for the picoamp current measurement (Suchyna, Markin et al. 2009).In contrast, bovine serum albumin (BSA) is used in our assay to minimize the nonspecific interaction between the aspirated cell and the glass (Fillafer, Mussel et al. 2018).
Thereby, the adhesion energy between the membrane and the glass was minimized, and only the physical constriction induced by the narrow geometry and the mechanical force applied by the aspiration would stimulate the cell.
In addition, we established a pulling protocol for a micropipette with new geometry to further explore how geometrical constriction from the narrow space would mediate the cellular mechano-response.Specifically, we post-processed the pulled micropipette with fire polish.
First, we fabricated a parallel micropipette with an inner diameter of 3.5 μm.Then, the cut micropipette was mounted to the MicroForge to slowly melt the glass wall at the tip to decrease the inner diameter.After the inner diameter decreased to 1 μm, the heat was removed to cool and solidify the shape.As a representative outcome, a micropipette with an inner diameter d = 1.12 μm at the tip was achieved (Fig. R5a), which exhibited a similar geometry at the tip compared to the normal micropipette with tip angle θ = 0° (Fig. 1c in the revised manuscript).
In sharp contrast, the inner diameter rapidly increased to d = 3.47 μm after the tip region.
When this new holding micropipette geometry aspirated the RBC, the calcium intensity change increased rapidly, particularly in the body region (Fig. R5b).Remarkably, the intensity change is higher in holding micropipette aspirated RBC (Fig. R5c; 7.17±1.59fold at ∆p = -25 mmHg) than in normal θ = 0° micropipette.Combining with our findings in the revised manuscript, we suggest that changing the later region diameter would upregulate the tension in the aspirated RBC, and therefore promote the PIEZO1-mediated calcium mobilization.However, such micropipette geometry is out of the scope of this study.Thus, we will not discuss it further in the revised manuscript.On the other hand, further investigating the gating mechanism of PIEZO1 at the molecular scale is not the focus of this study.Instead, we performed studies to investigate how the structure of F-actin would alter its accumulation at the micropipette tip (Fig. 5d in the revised manuscript), in terms of actin length (Latrunculin A, inhibiting actin monomer binding into Factin filament), F-actin branching (CK-666, inhibiting Arp2/3), and F-actin structure reinforcement (jasplakinolide, inducing actin polymerization).Importantly, our findings did not support that F-actin would directly engage with the PIEZO1 to modify the mechanical force transduction towards the channel, but rather proposed that accumulation would alter the mechanical transduction across the whole cell membrane, further enhance the mechanical tension and promote PIEZO1 activities explained by the 'force-from-lipid' model (Bavi, Nakayama et al. 2014).
Furthermore, in addition to changes in the local membrane tension, Piezo gating is also affected by the local membrane geometry---see, e.g., eLife 7 ( 2018): e41968---and it is not clear whether the results in the present manuscript are due to changes in the local membrane tension (as suggested by the authors) or due to the local membrane geometry (or a combination of both).

Response:
We are thankful for the reviewer providing this inspiring information.Despite the eLife 7 (2018): e41968, other studies have also reported that local membrane geometries (Haselwandter and MacKinnon 2018), channel density and cluster (Lewis and Grandl 2021), and resting membrane tension (Lewis and Grandl 2015) may also tune the channel gating.
However, since our focus is not delving into the gating mechanism of the PIEZO1 channels, local membrane geometry at the nanometer scale and its effect on PIEZO1 gating will not be further explored in this study.
Action taken: Neverthless, we have cited these works and included the discussion of this nanoscale geometry effect on PIEZO1-mediated mechano-response in the Discussion of the revised manuscript as stated in the previous response.
Page 20 Line 27 -33 "Studies have reported that PIEZO1 activities are not only mediated by the local membrane tension (Bavi, Cox et al. 2023), but also influenced by the local membrane footprint, or membrane curvature (Haselwandter andMacKinnon 2018, Young, Lewis et al. 2022) More importantly, the resting membrane tension before any mechanical stimulation (Lewis and Grandl 2015), the cluster and density of the channels (Lewis and Grandl 2021) can also alters the sensitivity and activities of the channels.Therefore, it is also important to take these factors into consideration in the further study when untacking the microgeometry effect on single cell mechanosensing." -Especially in the discussion section, the manuscript makes strong claims that are not sufficiently substantiated by the data or the modeling, given their very qualitative nature, thus overselling the results of this study.Moreover, some of the language used here, starting with the term "buckle" in the title, is rather sloppy; "Buckling" has a well-defined meaning in science, and the paper does not provide much evidence that this is indeed what is going on here.
Response: We thank the reviewer for pointing out the misleading wording in the manuscript.
Action take: We replaced all "buckle" with "F-actin accumulation" or "physical constriction".Combining this approach with computational analysis to calculate tension changes at the deformed membranes, the authors find that local Piezo1 responses scale with the pipette diameter and tip angle.Moreover, they discover a rapid actin redistribution into a buckle in the region of the cell right outside the pipette tip.This redistribution is in part Piezo1-dependent and promotes further channel activation.Importantly, contractility is dispensable for actin buckle amplification of Piezo1 function, pointing at actin accumulation as a mechanical input by itself.
The article is clearly written and illustrated, and its solid approach tackles an important issue regarding a channel family attracting a lot of attention in recent years: how are these channels activated by the cell environment?These findings seem especially relevant for RBC deformation by vessels and downstream volume regulation, known to depend heavily on Piezo1.However, the same could be said for white blood cells, and metastatic cancer cells both during blood-borne dispersion or invasion.In addition, one of the answers of this paper has relevant consequences for us Piezo researchers: we need to consider how we are polishing and holding our patch clamp pipettes in our daily experiments.These examples signify the broad relevance of this work Overall, I think this paper should be published after addressing some comments.

Response:
We are grateful for Reviewer #2's positive comments.Please check our point-bypoint response below.
Minor comments • This is clearly out of the scope of this paper and should not affect any editorial decision.Just curiosity: do the authors have any experiment using GOF Piezo1 mutants known to control RBC volume and cause xerocytosis (Cahalan, eLife, 2015 and references therein)?Is their method capable of capturing these differences?Are the mutations shifting any of the curves: diameter, angle?What happens to actin in those cells?
Response: We thank the reviewer for such insightful questions and the opportunity to clarify our experimental approach regarding GOF PIEZO1 mutants.While our study does not include experiments with these mutants, we understand their potential impact on RBC volume regulation and membrane tension.Instead, we have used the PIEZO1 agonist YODA1 as a positive control to validate that all the calcium mobilization captured in this study is PIEZO1dependant (Fig. S2a in the revised manuscript).YODA1 treatment, on the other hand, can be interpreted as an approach to mimic the performance of PIEZO1 GOF phenotypes in terms of gating since the agonist is known to lower the gating mechanical threshold for the channel (Syeda, Xu et al. 2015, Wang, Chi et al. 2018, Botello-Smith, Jiang et al. 2019).Here, we put the tip angle θ = 0° as an example to show the comparison.It is clear that after 0.5 µM Yoda1 treatment, the Ca 2+ intensity changes were upregulated, particularly in the pressure regime Δp = -15 to -30 mmHg (Fig. R6).However, the fact that Piezo1 triggers its actin-based own amplifier seems very relevant and should be discussed more in detail.

Response:
We thank the reviewer for this constructive suggestion.In fact, the finding we stated in this study cannot provide solid evidence that this is a linking mechanism between PIEZO1 and F-actin.In fact, we believe that F-actin accumulation at the neck region (i.e., part of the aspirated cell at the micropipette tip) serves as a physical constriction to the membrane environment.Such constriction would change the membrane tension at the tongue to the dome region of the aspirated cell, and thus, mediate PIEZO1 ion channel activities by following the 'force-from-lipid' model.To this end, we utilized the CK666 and other F-actin targeting treatments to modify the structure and thereby modify the impact of the physical constriction to prove that the strength of the F-actin constriction is correlated with the amplification of PIEZO1 activities at the tongue region.
Regarding the driving factor of F-actin accumulation, Luo et al. (Luo, Mohan et al. 2013) has reported that the shear force inside the cell under aspiration would accumulate the actin filaments at the neck region.Our finding of increasing aspiration pressure promoted the chance of observing F-actin accumulation at the neck further supports the reported finding (Figs.4d-f in the revised manuscript).Intriguingly, higher PIEZO1 expression level also favored the Factin accumulation in our results but delving into details to prove that such upregulation is solely depended on the PIEZO1 channels and their activities, or upregulation is also mediated by the expression level-induced mechanical property change in the cell line is out of the scope of this study.
• Figure 1A shows nucleated cells and RBCs in the cross-section of the vessel (top right) but seems to focus on nucleated cells in the longitudinal cut (bottom left).However, most experiments use RBCs (it's one of the paper keywords!).Just showing RBCs would make things clearer.

Response:
We appreciate the attention to detail in our figures.We agree that consistency in the cellular focus of our images is important for clarity.To avoid confusion and maintain focus on the primary cell type used in our experiments, we have revised Figure 1A to exclusively show RBCs in both the cross-section and longitudinal views of the vessel.
Action taken: The nucleated cell in Fig. 1a has been replaced with RBC.
• Line 240-241: If I got this right, which further substantiating should read which further substantiated or suppress which.
Action taken: Page 12 Line 10 "substantiating" has been replaced with "substantiated" • Line 244: accumulates should read accumulate Action taken: Page 14 Line 7 "accumulates" has been replaced with "accumulate" • Line 254: accumulation formation sounds redundant to me.Accumulation should be enough.
Action taken: Page 14 Line 17 and Line 19 "accumulation formation" has been replaced with "accumulation" • I think the title of Fig. S3 needs rewriting.

Action taken:
The title of Fig. S3 has been rewritten for accuracy and precision, now titled "Piezo1-KO RBC suppressed calcium mobilization in aspirated RBCs" more pronounced F-actin accumulation … in PIEZO1-WT and PIEZO1-OE genotypes (Fig. S8a) whereas absence of the mechanosensitive ion channel in PIEZO1-KO genotype leads to few accumulation events recorded in this study and the trend is not clear.Nevertheless, results suggest that…" Reviewer #3 (Remarks to the Author): The author describes a fluorescent micropipette aspiration assay for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time and FLIM analysis was used.The idea is novel and the findings lead to future biomedical applications.
Minor comments-Authors have shown F-actin mobility modulates PIEZO1 activation at aspirated cell tongue where HEK293T cells are used.How this analysis corelates with the local membrane tension as shown for RBC based results?
Response: The FLIM measurement on RBC proves the microscale geometrical modulation on local membrane tension, showing that higher tip angles (i.e., θ = 10°) would facilitate a higher membrane tension at the aspirated tongue.Meanwhile, this higher tension was further amplified by the physical constriction after F-actin accumulating at the aspirated neck region.
Thereby, our results demonstrated a further strengthened PIEZO1 activities at the tongue.
What are the excitation and emission wavelengths filters used for fluorescence signal measurement ?
Action taken: Information has been added to the Method section: Page 32 Line 14 -Line 15: "A filter cube with 475/28 nm excitation filter, 561 nm dichroic cut-off mirror, and 609/57 nm emission filter was in place to separate fluorescence." What was the acquisition time for single FLIM image ?

Action taken: Information has been added
Page 32 Line 11 -Line 13: "To ensure enough photon counting for lifetime analysis, temporal resolution was sacrificed and a total number of 5 frames (total acquisition time = 20 s) were stacked for each acquisition." What are the challenges of FLIM measurement in this study?
Response: There are two major challenges in FLIM measurements: \

Figure 1 -
Updated to include a red blood cell, aligning with reviewer feedback.o Figure 3 -Enhanced with new simulation data from elastic shell FEA study and quantitative membrane tension analysis.o Figure 5 -Title modified to "F-actin accumulation influences PIEZO1 activity within the cellular protrusion."o Figure S3 -Caption revised to "Calcium

Figure R1 (
Figure R1 (revised Figure S13).Modelling red blood cell deformation upon micropipette aspiration.a Initial setup for the RBC aspiration modeling, displaying the undeformed biconcave geometry of a real RBC as used in the computational simulations.The undeformed state serves as a baseline for subsequent deformation analysis.b Snapshots of RBC aspirated

Figure R2 (
Figure R2 (revised Figure 3f-h).a-c Front view of the FEA simulated aspirated RBC maximum principal stress contours, denoted the change of membrane tension distribution on the aspirated RBCs.Values are normalized based on the maximum principal stress value of all conditions.Black arrow pointed to the regions with the highest membrane tension when the cell was aspirated by θ = 0° (a) 5°(b) and 10°(c).

Figure R3 (
Figure R3 (revised Figure 3 a-e) Membrane tension mapping of RBC aspirated by micropipettes with different tip angles.a-c, top panel: Representative fluorescence lifetime mapping of individual RBCs labelled with Flipper-TR and aspirated at ∆p = -25 mmHg.The RBCs were aspirated by micropipettes with θ = 0° (a), 5° (b) and 10° (c), respectively.bottom panel: Fluorescence lifetime distribution of aspirated cell body (orange) and tongue (red) regions were plotted and fitted with Gaussian distribution.d, statistics of Flipper-TR lifetime of RBCs aspirated by micropipettes with different angles.The lifetime of the cell body and tongue regions were quantified separately.A major increase in the lifetime was noticed in both body and tongue regions when the tip angle θ increased.e the ratio of tongue-to-body lifetime . 4b; r = 0.99).Since it has been reported that increasing the membrane tension by ~3-fold would facilitate PIEZO1 from limited gating to fully gating(Cox, Bae et al. 2016), the change of tension detected by the FLIM assay here further demonstrates the statement we made in the original manuscript -"increasing tip angle would be the major cause of the elevated calcium influx during aspiration, especially in the tongue region.".

Figure R4 (
Figure R4 (revised Figure S5) Correlation Between PIEZO1 Activation versus Membrane Tension, and Simulated Stress versus Membrane Tension.Pearson correlation tests was performed between FLIM versus fMPA (a) and FEA versus FLIM (b) results at ∆p = -25mmHg.Statistical outcome demonstrated a strong positive correlation between FLIM versus fMPA results (r = 0.83) and FEA versus FLIM results (r = 0.99).The correlation underscores our hypothesis that tip angle variation is a key mechanical factor influencing PIEZO1 channel activity, further supported by FLIM data and FEA results demonstrating the tension increase with larger tip angles.
It has been known for some time that Piezo gating is affected by force on the membrane/the channel and cytoskeletal attachments(e.g., ref. 26  in the manuscript and NatureCommunications 7.1 (2016): 12939---a paper that should be discussed in this context).The present manuscript adds a twist to this, by putting emphasis on the micropipette shape.To gain new, solid understanding of Piezo gating in cell membranes, quantitative data on the molecular mechanisms underlying the observed behavior would be required.For instance, there are many ways in which the micropipette might affect membrane tension---e.g., through the physics of membrane-pipette adhesion, the constrained geometry at the tip of the micropipette, patchclamp induced changes in membrane organization, etc.Response: Thank you for highlighting the intricate factors influencing Piezo gating.Our study indeed brings attention to the micropipette shape, adding a novel perspective to the mechanical environment's role in channel gating.We acknowledge the complexity of micropipettemembrane interactions and their potential to affect membrane tension through various mechanisms.While our current data does not dissect these mechanisms in detail, it provides a foundational understanding of the geometric influence on PIEZO1 activation.

Figure R5 .
Figure R5.RBC calcium mobilization when aspirated by a holding micropipette with modified geometry.a new pulling protocol helps fabricate a holding micropipette with a narrow opening.After cutting the micropipette to obtain an inner diameter of 3.5 μm, the micropipette was placed on a microforge to perform fire polishing.The micropipette was placed next to the heating source and the glass wall was melted to narrow down the tip slowly.The heat was stopped once the tip diameter decreased to 1 μm.As shown in the representative DIC image, the tip diameter is 1.12 μm.b Representative fluorescence snapshot of holding micropipetteaspirated RBC.c, Ca 2+ intensity change at different aspiration pressure when the RBC is aspirated by holding a micropipette.As the pressure increases, the intensity change in the RBC aspirated by the holding pipette was larger than the RBC aspirated by a normal parallel micropipette with tip diameter d = 1 μm.Intensity fold change was measured from n ≥ 6 aspirated RBCs to obtain mean ± s.e.m.

Reviewer # 2 (
Also, the paper starts off by talking about the binding site-driven or membrane curvaturedriven Piezo localization described in refs.19, 20 in the manuscript, while the manuscript argues (based on somewhat flimsy evidence) that the observed effects are driven by changes in the local membrane tension.This is confusing, as membrane geometry, Piezo binding, and local changes in membrane tension provide distinct physical mechanisms potentially affecting Piezo gating (see also the point above).Response: We agreed that talking about the impact of curvature on PIEZO1 in the Introduction may give readers misleading information.Action taken: We moved the section to the Discussion.Remarks to the Author): Summary: In this manuscript, Wang et al develop combine multi-channel imaging and micropipette aspiration to study how local membrane deformation activates the mechanically activated ion channel Piezo1.

Figure R6 .
Figure R6.Yoda1-dependent Ca 2+ mobilization on aspirated RBC change with different aspiration pressures.Intensity fold change was measured from n ≥ 6 aspirated RBCs to obtain mean ± s.e.m..

and Discussion Enhancement: "
 Conclusion